We thank Drs. Martin M. Schmitt and Line Fraemohs. This work was supported by the Netherlands Foundation for Scientific Research (ZonMW VIDI 016.126.358, the Landsteiner Foundation for Blood Transfusion Research (LSBR Nr. 1638) and Deutsche Forschungsgemeinschaft (SFB1123/A2) awarded to R.R.K.

All methods described here have been approved by the Medical Ethical and Animal Ethical Boards of Maastricht University.
1. Flow-Based Assay with Human Cells
<ol>
	<li>Isolation of platelets from human blood
	<ol>
		<li>Draw venous blood in citrate (3.2%) anticoagulant.</li>
		<li>Add 1/15 volume of Acid Citrate Dextrose (ACD: 80 mM trisodium citrate, 52 mM citric acid and 183 mM glucose) to the blood.</li>
		<li>Centrifuge at 350 x g without brake for 15 min to obtain plate...

<ol>
	<li>Dinauer, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+immune+deficiencies+with+defects+in+neutrophil+function.">Primary immune deficiencies with defects in neutrophil function.</a> American Society of Hematology. 2016 (1), 43-50 (2016).</li><li>Butcher, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte-endothelial+cell+recognition:+three+(or+more)+steps+to+specificity+and+diversity.">Leukocyte-endothelial cell recognition: three (or more) steps to specificity and diversity.</a> Cell. 67 (6), 1033-1036 (1991).</li><li>Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traffic+signals+for+lymphocyte+recirculation+and+leukocyte+emigration:+the+multistep+paradigm.">Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm.</a> Cell. 76 (2), 301-314 (1994).</li><li>Nourshargh, S., Alon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+migration+into+inflamed+tissues.">Leukocyte migration into inflamed tissues.</a> Immunity. 41 (5), 694-707 (2014).</li><li>Legein, B., Temmerman, L., Biessen, E. A. L., Lutgens, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+and+immune+system+interactions+in+atherosclerosis.">Inflammation and immune system interactions in atherosclerosis.</a> Cellular and Molecular Life Sciences. 70 (20), 3847-3869 (2013).</li><li>Wang, Y., Sakuma, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+engagement+of+platelet+glycoprotein+Ibalpha+via+the+integrin+Mac-1+is+critical+for+the+biological+response+to+vascular+injury.">Leukocyte engagement of platelet glycoprotein Ibalpha via the integrin Mac-1 is critical for the biological response to vascular injury.</a> Circulation. 112 (19), 2993-3000 (2005).</li><li>Zhao, Z., Vajen, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+junctional+adhesion+molecule+A+from+platelets+increases+early-stage+neointima+formation+after+wire+injury+in+hyperlipidemic+mice.">Deletion of junctional adhesion molecule A from platelets increases early-stage neointima formation after wire injury in hyperlipidemic mice.</a> Journal of cellular and molecular medicine. , (2017).</li><li>Wang, Y., Gao, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+integrin+Mac-1+regulates+thrombosis+via+interaction+with+platelet+GPIb&#945;.">Leukocyte integrin Mac-1 regulates thrombosis via interaction with platelet GPIb&#945;.</a> Nature communications. 8, 15559 (2017).</li><li>Fujii, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDMS-based+microfluidic+devices+for+biomedical+applications.">PDMS-based microfluidic devices for biomedical applications.</a> Microelectronic Engineering. 61-62, 907-914 (2002).</li><li>Son, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+shear+viscosity+and+shear+rate+from+pressure+drop+and+flow+rate+relationship+in+a+rectangular+channel.">Determination of shear viscosity and shear rate from pressure drop and flow rate relationship in a rectangular channel.</a> Polymer. 48 (2), 632-637 (2007).</li><li>Brinkmann, V., Laube, B., Abu Abed, U., Goosmann, C., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps:+how+to+generate+and+visualize+them.">Neutrophil extracellular traps: how to generate and visualize them.</a> Journal of visualized experiments : JoVE. (36), e1724 (2010).</li><li>Swamydas, M., Lionakis, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+purification+and+labeling+of+mouse+bone+marrow+neutrophils+for+functional+studies+and+adoptive+transfer+experiments.">Isolation, purification and labeling of mouse bone marrow neutrophils for functional studies and adoptive transfer experiments.</a> Journal of visualized experiments : JoVE. (77), e50586 (2013).</li><li>Schmitt, M. M. N., Megens, R. T. 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This in vitro assay is a straightforward method to investigate underlying mechanisms of leukocyte recruitment during vascular inflammation, but there are some critical points to be noted. The first requirement for successfully performing this assay is the perfusion of leukocytes over an intact and confluent vascular or platelet monolayer. This can be achieved by prior coating of the surfaces with collagen type I. In general, when working with primary vascular cells, it is important to gently detach cells using t...

Upon injury, inflammation, or infection, leukocytes quickly respond to pathogen- or damage-associated molecular patterns (PAMPs, DAMPs), change into an activated state, and move out of the blood stream to sites of inflammation and tissue damage. The ability of leukocytes to interact with their cellular and molecular environment is essential for their correct function as immune cells, as highlighted by genetic disorders such as leukocyte adhesion deficiency1. Leukocyte adhesion has been the subject of intense investigation during the past decades and this has resulted in the concept of the leukocyte adhesion cascade in the early 1990s2,3. Leukocyte adhesion is initiated by the selectin-mediated capture of leukocytes to the endothelium, causing the cells to roll over the vascular surface. This rolling enables leukocytes to scan for endothelium-bound migratory cues, e.g., chemokines, which induce the activation of integrins. Subsequently, the activated integrins mediate the binding to endothelial ligands, resulting in firm leukocyte arrest. Leukocytes may subsequently prepare to extravasate by crawling and spreading, before penetrating the endothelial monolayer and transmigrating into the underlying tissue. The basic concept of the canonical leukocyte cascade has remained largely unchanged since its introduction, with some intermediate steps added4. Nevertheless, the exact molecular mechanisms and the roles of the many players involved in leukocyte recruitment have not been clarified thus far, and leukocyte recruitment remains an important subject in the field of infection, inflammation, and (auto-) immune research.
For example, during vascular inflammatory diseases such as atherosclerosis, increased leukocyte recruitment into the vessel wall drives plaque development. Unstable atherosclerotic plaques might rupture, leading to massive activation of platelets and the coagulation system, and subsequently to occlusion of the vessel5. This may result in severe cardiovascular outcomes such as myocardial infarction or stroke. In addition, endothelial denudation as it occurs clinically, e.g. after stenting of a coronary artery, leads to a multitude of interactions of leukocytes and platelets to the exposed vessel wall interior (e.g., matrix components and smooth muscle cells) and of leukocytes with platelets covering the vascular injury. These interactions are important for the further development of the disease as monocyte-platelet interactions might drive neointima formation6,7. In addition, platelet-leukocyte interactions mediated by leukocyte integrin Mac-1 (&#945;M&#946;2) and platelet GPIb&#945;&#160;have recently been identified as novel drivers of thrombosis in mice8.
Given the wide availability of human and animal blood as a source of leukocytes and platelets for research, and the broad spectrum of isolated matrix molecules and immortalized cell lines of leukocyte and vascular origin, it is feasible to simulate leukocyte interactions under flow in a laboratory setting, using specially designed flow perfusion chambers. Many variants have been designed over the past decades, ranging from vacuum-driven to self-adhesive perfusion chambers. All variants have in common that the immobile part (e.g., cultured vascular cells or matrix proteins) is assembled into a larger leak-proof chamber equipped with a pre-defined channel enabling perfusion of fluids over the immobile part. In addition, advances in molding technology enabled the development of custom-made solutions based on silica polymers9. The viscosity and flow rate of the perfused fluids and the height of the channel mainly determine the shear stress characteristics of the flow perfusion device10. In this article, we present an in vitro method to study underlying mechanisms of the adhesion, de-adhesion, and transmigration of leukocytes under venous and arterial flow regimes. The advantage of the methods presented here is that they can be performed using a common camera-connected fluorescence microscope, and do not require a experimenters to possess high technical proficiency. The in vitro assay can be manipulated in many ways (e.g., by adding inhibitors or blocking antibodies), and is thus applicable in different models of vascular inflammation and allows the investigation of adhesion protein functions or the evaluation of specific compounds.

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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Inverted fluorescence microscope e.g. EVOS-FL</td>
			<td style="width:189px;">Life Technologies Europe bv</td>
			<td style="width:163px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Pump e.g. model: 210-CE</td>
			<td style="width:189px;">&#160;world precision instruments</td>
			<td style="width:163px;">78-9210W</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Falcon 35 mm TC-Treated Easy-Grip Style Cell Culture Dish</td>
			<td style="width:189px;">corning</td>
			<td style="width:163px;">353001</td>
			<td></td>
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			<td height="20" style="height:20px;width:420px;">50 mL syringe</td>
			<td style="width:189px;">Becton Dickinson</td>
			<td style="width:163px;">300137</td>
			<td></td>
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			<td height="20" style="height:20px;width:420px;">Silicone tubing&#160;</td>
			<td style="width:189px;">VWR</td>
			<td style="width:163px;">228-0700</td>
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			<td height="20" style="height:20px;width:420px;">Elbow Luer Connector Male</td>
			<td style="width:189px;">Ibidi</td>
			<td style="width:163px;">10802</td>
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			<td height="20" style="height:20px;width:420px;">Female Luer Lock Coupler</td>
			<td style="width:189px;">Ibidi</td>
			<td style="width:163px;">10823</td>
			<td></td>
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			<td height="20" style="height:20px;width:420px;">Flow chamber</td>
			<td style="width:189px;">University Hospital RWTH Aachen</td>
			<td style="width:163px;"></td>
			<td>Patent DE10328277A1: Baltus T, Dautzenberg R, Weber CPD. Str&#246;mungskammer zur in-vitro-Untersuchung von physiologischen Vorg&#228;ngen an Zelloberfl&#228;chen. 2005.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Ibidi sticky slide VI 0.4</td>
			<td style="width:189px;">Ibidi</td>
			<td>80608</td>
			<td></td>
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			<td height="20" style="height:20px;">Coverslips for sticky slides&#160;</td>
			<td style="width:189px;">Ibidi</td>
			<td>10812</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagen Type I, rat tail</td>
			<td>Life Technologies Europe bv</td>
			<td style="width:163px;">A1048301</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Recombinant Human TNF-&#945;</td>
			<td>Peprotech</td>
			<td>300-01A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thrombin Receptor Activator for Peptide 6 (TRAP - 6)</td>
			<td>Anaspec / Eurogentec</td>
			<td>24191</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SYTO 13 green fluorescent nucleic acid stain</td>
			<td>Life Technologies Europe bv</td>
			<td>S7575</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Hanks&#8217; Balanced Salt Solution 10x</td>
			<td>Sigma-Aldrich Chemie</td>
			<td>H4641</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES buffer solution 1M, liquid</td>
			<td>Life Technologies Europe bv</td>
			<td>15630049</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BUMINATE Albumin, 25%&#160;</td>
			<td>Baxter</td>
			<td>60010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Water for injection</td>
			<td style="width:189px;">B. Braun</td>
			<td style="width:163px;">3624315</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Calcium chloride dihydrate</td>
			<td style="width:189px;">Merck</td>
			<td style="width:163px;">102382&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Magnesium chloride hexahydrate&#160;</td>
			<td>Sigma-Aldrich Chemie</td>
			<td style="width:163px;">M2670</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Apyrase</td>
			<td>Sigma-Aldrich Chemie</td>
			<td>A6535</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Primary Human Umbilical Vein Endothelial Cells (HUVEC)</td>
			<td style="width:189px;">Promocell GmbH</td>
			<td>C-12203</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Endothelial Cell Growth Medium (Ready-to-use)</td>
			<td style="width:189px;">Promocell GmbH</td>
			<td>C-22010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Primary Human Aortic Endothelial Cells (HAoEC)</td>
			<td style="width:189px;">ATCC</td>
			<td style="width:163px;">&#160;PCS-100-011</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Vascular Cell Basal Medium&#160;</td>
			<td style="width:189px;">ATCC</td>
			<td style="width:163px;">PCS-100-030</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Endothelial Cell Growth Kit-BBE</td>
			<td style="width:189px;">ATCC</td>
			<td style="width:163px;">PCS-100-040</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Primary Human Aortic Smooth Muscle Cells (HAoSMC)</td>
			<td style="width:189px;">ATCC</td>
			<td style="width:163px;">PCS-100-012</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Vascular Smooth Muscle Cell Growth Kit</td>
			<td style="width:189px;">ATCC</td>
			<td style="width:163px;">PCS-100-042</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Human endothelial cell line, EA.hy926</td>
			<td style="width:189px;">ATCC</td>
			<td style="width:163px;">CRL-2922</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td style="width:189px;">ATCC</td>
			<td style="width:163px;">30-2002</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Mouse endothelial cell line, SV 40 transformed (SVEC4-10)</td>
			<td style="width:189px;">ATCC</td>
			<td style="width:163px;">CRL-2181</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Human Monocytic cell line, THP-1</td>
			<td style="width:189px;">DSMZ</td>
			<td style="width:163px;">ACC-16</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI 1640 with Ultra-Glutamine</td>
			<td style="width:189px;">Lonza</td>
			<td>BE12-702F/U1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Mouse monocyte/macrophage RAW264.7</td>
			<td style="width:189px;">ATCC</td>
			<td style="width:163px;">TIB-71</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM), high glucose</td>
			<td style="width:189px;">Gibco</td>
			<td style="width:163px;">11965092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Accutase</td>
			<td style="width:189px;">Promocell GmbH</td>
			<td>C-41310</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Percoll</td>
			<td style="width:189px;">Sigma-Aldrich Chemie</td>
			<td>P1644</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Histopaque 1119</td>
			<td style="width:189px;">Sigma-Aldrich Chemie</td>
			<td>11191</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:420px;">10x PBS</td>
			<td style="width:189px;">Life Technologies Europe bv</td>
			<td style="width:163px;">14200075</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:420px;">BD&#160;Vacutainer Plastic Blood Collection Tubes with Sodium Heparin</td>
			<td style="width:189px;">Becton Dickinson</td>
			<td style="width:163px;">367876</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">VACUETTE TUBE 9 ml 9NC Coagulation sodium citrate 3.2%&#160;</td>
			<td style="width:189px;">Greiner Bio</td>
			<td style="width:163px;">455322</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:420px;">HCl/EtOH mixture (1.2 mol/L HCl and 50% ethanol) in water</td>
			<td style="width:189px;">Sigma-Aldrich Chemie</td>
			<td style="width:163px;">Prepare by mixing 0.3L HCl (4 mol/L) with 0.7L of 70% v/v ethanol in a fume hood</td>
			<td></td>
		</tr>
	</tbody>
</table>

laminar flow-based assays to investigate leukocyte recruitment on cultured vascular cells and adherent platelets

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted in the concept of the leukocyte adhesion cascade. However, the exact molecular mechanisms involved in leukocyte recruitment have not yet been fully identified. Since leukocyte recruitment remains an important subject in the field of infection, inflammation, and (auto-) immune research, we present a straightforward laminar flow-based assay to study underlying mechanisms of the adhesion, de-adhesion, and transmigration of leukocytes under venous and arterial flow regimes. The in vitro assay can be used to study the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners in different models of vascular inflammation. This protocol describes a laminar flow-based assay using a parallel-flow chamber and an inverted phase contrast microscope connected to a camera to study the interactions of leukocytes and endothelial cells or platelets, which can be visualized and recorded then analyzed offline. Endothelial cells, platelets, or leukocytes can be pretreated with inhibitors or antibodies to determine the role of specific molecules during this process. Shear conditions, i.e. arterial or venous shear stress, can be easily adapted by the viscosity and flow rate of the perfused fluids and the height of the channel.

To study endothelial-leukocyte adhesion, fluorescently labeled THP-1 cells were perfused over a TNF&#945;- or non-stimulated endothelial monolayer for 2 min at 3 dyne/cm2. The total number of adherent monocytic cells was determined after 2 min of perfusion by capturing 6 independent fields over a period of 2-6 min. The adherent cells were quantified in at least 6 pictures captured with an inverted phase contrast/fluorescence microscope (e.g., EVOS-FL) using 10X magnifi...

Watch this Scientific Journal Video about Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets at JoVE.com

Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets

Leukocytes avidly interact with vascular cells and platelets after vessel wall injury or during inflammation. Here, we describe a straightforward laminar flow-based assay to characterize the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners.

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted ...

The overall goal of this experiment is to investigate the mechanisms behind the interactions of leukocytes to adherent vascular cells or platelets under flow conditions. This method can help answer key questions in the immunology field such as how leukocytes are recruited to sites of inflammation. The main advantage of this technique is that it's cost effective, user friendly, and allows the investigation of a great variety of experimental conditions.This method can provide insight into leukocyte adhesion. It can also be applied to other systems such as the adhesion of tumor cells to immobilized cells or substrates under flow conditions. Demonstrating the procedure will be Annemiek Dickhout, an engineer and a PhD student working in my laboratory.After obtaining blood samples, add 1/15th volume of acid citrate dextrose to the blood. Next, centrifuge the blood sample by setting the brakes off at 350 times g for 15 minutes to obtain platelet-rich plasma. Then transfer the supernatant obtained to a 15 milliliter conical tube and add 1/10th volume of acid citrate dextrose to it.Again, centrifuge the suspension by setting the brakes off at 1, 120 times g for 15 minutes to obtain platelet-poor plasma. Next, discard the supernatant. Next, carefully resuspend the platelet pellet in platelet buffer maintained at pH 6.6 using a Pasteur pipette with a wide bore.Then add 1/10th volume of acid citrate dextrose to it and gently mix it. Next, centrifuge the platelets at 1, 120 times g for 15 minutes to form the pellet. After the centrifugation is over, discard the supernatant and resuspend the platelets in one milliliter of platelet buffer maintained at pH 7.5.Then use an automated hematology analyzer to measure the platelet concentration in 1/10th dilution. Finally, adjust the cell count with platelet buffer maintained at pH 7.5. To secure the adherent platelets, immerse the glass coverslip in 1.2 molar 50%volume by volume of hydrochloric acid ethanol mixture.After immersing, wash the coverslip twice with ultrapure water. Then use nitrogen to dry the coverslip. Next, use 100 microliters of 30 micrograms per milliliter of rat collagen type one to pre-coat the glass coverslip.After coating the glass coverslip, incubate the coverslips at room temperature for 30 minutes. Then expel the collagen solution and wash the coverslip with phosphate buffered saline thrice. Next, use 1%bovine serum albumin in HEPES buffer to block the collagen coated coverslips for 30 minutes at room temperature.After 30 minutes, wash the coverslips and then dry them to mount in the perfusion chamber. After mounting the coverslips, keep the platelets ready. To immobilize the platelets, transfer 70 microliters of platelet suspension to a channel and incubate for 1-1/2 hours at 37 degrees Celsius and 5%carbon dioxide.Then replace the nonadherent platelets with blocking buffer for 30 minutes at room temperature. Use one micromolar of cell-permeant green fluorescence nucleic acid stain to fluorescently label the leukocytes. After labeling, incubate the leukocytes for 30 minutes at 37 degrees Celsius.Then centrifuge the cells at 300 times g for five minutes to pellet the cells. Resuspend in phosphate buffered saline to wash the cells. Then centrifuge the cells at 300 times g for five minutes.Based on the flow rate, suspend five milliliters of the cells in assay buffer. Then heat a water bath to maintain 37 degrees Celsius. Next, set aside a 50 milliliter perfusion syringe and install it on the syringe holder.Then set the pump on with draw mode with the pump volume at zero milliliter and diameter at 26.70 millimeter. Next, attach a Luer lock coupler to the syringe and connect the tubing with the elbow Luer connector to the Luer lock coupler. Then attach the free end of the tubing to the flow chamber.To adhere the leukocytes to the immobilized platelets, connect the microslide with the syringe. To perfuse the leukocytes and then adhere to a bed of vascular or platelet monolayer, attach a second tubing to a 50 milliliter conical tube containing the assay buffer and then fill the tubing using a one milliliter pipette. Once the tubing is filled, squeeze it and connect it to the flow chamber.Before perfusing the leukocytes, add three millimolar of calcium chloride and two millimolar of magnesium chloride to the cell suspension. Then incubate the cells at 37 degrees Celsius for five minutes. To remove possible air stuck in the chamber and in the tubing, perfuse with assay buffer before perfusing the cells.Next, squeeze the tubes to close the end and switch the tubing from the assay buffer to the cell suspension thereby preventing any trapped air bubbles within. For the first cells to arrive, perfuse the cells at appropriate flow rate and shear stress. Keep perfusing the cells, maintaining the flow rate and shear stress for two minutes.Then capture six images of the rolling and adherent cells between two to six minutes using a fluorescence microscope with 10X magnification connected to a digital camera. In this assay, adhesion of green fluorescently labeled THP1 cells with the endothelial cells was analyzed in the presence or absence of tumor necrosis vector alpha stimulation. The graph and the fluorescence images obtained show significant increase in THP1 cell arrest upon stimulating the endothelial cells with tumor necrosis vector alpha in comparison to the control.When recorded in time lapse, transendothelial migration of primary monocytes can be monitored over time. Next, adhesion of fluorescently labeled neutrophils with immobilized platelet monolayer in a flow chamber was analyzed in the presence or absence of TRAP-6 stimulation. The plot obtained shows a sharp increase in the adhesion of neutrophils to the TRAP-6 stimulated platelets in comparison to the control.Next, platelets were obtained from JAM-A deficient mice to study its adhesion with raw 264.7 mouse monocyte in the presence of surface receptor inhibitors. The graph and the fluorescence images obtained show significant decrease in the adhesion of the mouse monocytes with the platelets when compared to the control. Further, to study the adhesion of raw 264.7 cells with platelets, JAM-A positive and deficient mice were exposed to incremental increase of shear stress.From the graph, it's clear that the adhesion of raw 264.7 cells with platelets obtained from JAM-A deficient mice are more resistant to shear stress than JAM-A positive platelets. Once mastered, this technique can be done in one day if performed properly. While attempting this procedure, it is important to very gently wash your platelets during isolation so that your platelets do not become pre-activated or aggregated.It is also crucial to make sure that you don't have any air bubbles in your flow system because the system might get congested and to ensure a stable and even shear stress. Following this procedure, underlying mechanisms of leukocyte recruitment during vascular inflammation can be investigated.

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Research

JoVE Journal

Immunology and Infection

7.5K vistas.  Maastricht University. The overall goal of this experiment is to investigate the mechanisms behind the interactions of leukocytes to adherent vascular cells or platelets under flow conditions. This method can help answer key questions in the immunology field such as how leukocytes are recruited to sites of inflammation. The main advantage of this technique is that it's cost effective, user friendly, and allows the investigation of a great variety of experimental conditions.This method can provide insight int...